For environments such as oral cavities, in which a complex bacterial flora exists, it is difficult to describe the cause of diseases or the like by merely determining the presence or absence of a certain bacterium. Thus, the establishment of analysis systems that can identify bacterial flora patterns has been deemed to be desired.
In the analysis of a bacterial flora constituted by plural microorganisms, gene analysis methods such as clone library, FISH (Fluorescent In Situ Hybridization), DGGE (Denaturing Gradient Gel Electrophoresis), and T-RFLP (Terminal Restriction Fragment Length Polymorphism) methods have been employed in recent years. Of them, since the T-RFLP method provides for high resolution or sensitivity, convenient operation, and multi-sample processing in short time, the T-RFLP method is particularly useful for the rapid analysis of samples containing unknown bacterial species or many bacterial species.
Methods for analyzing a bacterial flora on the basis of 16S rRNA gene sequences using the T-RFLP method have been utilized in many fields such as industrial or medical fields, for example, wastewater treatment, bioremediation, and slime control (JP-A-2006-94830, JP-A-2006-25791, JP-A-2006-42817, and JP-A-2003-265199; Appl. Environ. Microbiol., 65 (8), 3518-25, 1999; Appl. Environ. Microbiol., 68 (4), 1854-63, 2002; Appl. Environ. Microbiol., 69 (2), 1251-62, 2003; and J. Med. Microbiol., 52, 79-89, 2003).
In the T-RFLP method, when using bacteria with already determined 16S rRNA gene sequences, the lengths of fragments cleaved with a restriction enzyme can be calculated as theoretical values by use of Ribosome Data Project II (RDPII), Microbial Community Analysis (MiCA), and the like. However, this method presented the problem of difficulty in assigning bacteria since the number of bases of samples measured with actual fragment-length measurement (capillary electrophoresis) deviates from the theoretical values. Thus, an approach has been adopted in many cases in which the T-RFLP method is carried out simultaneously with the clone library method and compared therewith to thereby assign bacterial species corresponding to peaks. However, this approach requires enormous cost and efforts for conducting analysis using many samples.
Moreover, bacteria accidentally having the same lengths of 5′-terminal cleaved fragments cannot be distinguished by single restriction enzyme treatment. Therefore, this method presented the problem that plural restriction enzyme treatments must be done.